The researchers randomly assigned 65 piglets, 28 days old and weighing 7.59 ± 1.11 kilograms, to five treatments with 13 animals per group. The groups received a basal control diet, a diet supplemented with 1% L-glutamate, a diet supplemented with 1% L-aspartate, a diet containing 1% of each amino acid, or basal diet supplemented with 50 mg/kg carbadox. All pigs were orally inoculated with F18 ETEC for three consecutive days, beginning on day 7.

Blood was collected before inoculation and on days 9, 11, 21 and 28. The small intestine was sampled on day 12, near peak infection, and day 28, during recovery. The protocol was reviewed and approved by the UC Davis Institutional Animal Care and Use Committee under protocol 23529.

The challenge was followed by changes in blood-cell profiles and circulating markers of inflammation, although the response to dietary treatment was selective rather than uniform. White blood cell, neutrophil, lymphocyte, monocyte, eosinophil and basophil counts increased after inoculation and peaked on day 21, with no treatment differences in these populations at the individual sampling points. Red blood cell count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count and total protein concentration also increased on days 9 and 11 relative to pre-inoculation values, while hematocrit, mean corpuscular volume, red cell distribution width and mean platelet volume decreased.

Serum tumor necrosis factor-alpha rose on days 11, 21 and 28, serum interleukin-8 increased on day 11, and C-reactive protein and haptoglobin rose during the early post-challenge period. These infection-associated shifts occurred across the experiment, whereas the amino-acid groups differed from controls or the carbadox group in selected outcomes at particular times. Differences in red blood cell indices were most evident during recovery.

On day 21, piglets receiving L-glutamate or L-aspartate had higher hemoglobin concentration, hematocrit and mean corpuscular hemoglobin than controls. Mean corpuscular volume was also greater in the L-glutamate, L-aspartate and combined-supplement groups than in controls. At that point, L-glutamate and the combination had higher red cell distribution width than controls, while L-aspartate did not show the same elevation.

By day 28, L-glutamate and L-aspartate had lower red cell distribution width than controls, and L-aspartate also showed a tendency toward a greater mean corpuscular volume. These measurements document differences in red blood cell size, hemoglobin content and population uniformity. Systemic inflammatory markers showed a similarly time-dependent pattern.

On day 9, L-aspartate and the combined treatment had lower serum interleukin-8 concentrations than controls, while L-glutamate and L-aspartate had lower C-reactive protein than pigs receiving carbadox. On day 11, all three amino-acid treatments had lower C-reactive protein than controls and the antibiotic group, although pigs receiving the combined supplement had the highest serum tumor necrosis factor-alpha among the groups. By days 21 and 28, haptoglobin was substantially higher in controls than in each amino-acid group and in the carbadox group.

No treatment differences were detected for tumor necrosis factor-alpha, interleukin-8 or C-reactive protein on those later dates. The results therefore show modulation of selected inflammatory markers rather than broad or consistent suppression of every inflammatory signal. Mucosal gene-expression measurements revealed additional differences by intestinal segment and stage of infection.

On day 12, L-aspartate reduced ileal interleukin-8 expression compared with controls, the combined treatment and carbadox. In contrast, pigs receiving both amino acids had higher ileal interleukin-1 beta and interleukin-17A expression than pigs receiving L-aspartate or carbadox, as well as higher jejunal interleukin-1 beta expression. L-glutamate tended to have lower jejunal Toll-like receptor 4 expression, and the combination tended to have lower ileal tumor necrosis factor-alpha expression.

These results came from messenger RNA analysis of intestinal tissue. They establish differences in transcriptional responses, not direct evidence of altered cytokine protein abundance, bioactivity, epithelial integrity or clinical disease severity. By day 28, the intestinal expression profile included several genes associated with immune regulation and tissue repair.

In the jejunum, L-glutamate, L-aspartate and the combination tended to increase interleukin-4 expression relative to controls, while L-glutamate, L-aspartate and carbadox significantly increased interleukin-13 expression. In the ileum, the combined treatment had the greatest interleukin-4 expression, and L-glutamate had greater transforming growth factor beta 1 expression than the other treatments. Untargeted metabolomics offered an exploratory view of biochemical differences among the groups.

The researchers detected 414 features in day-12 jejunal mucosa, including 170 identified metabolites, and 393 features, including 161 identified metabolites, in day-12 ileal mucosa. Day-11 serum contained 402 detected features, of which 142 were identified. Higher jejunal aspartate abundance in pigs receiving L-glutamate or the combination met the predefined exploratory criteria, as did higher ileal glutaric acid in the combined group.

Large fold changes included increased serum palmitoleic and oleic acids with all three amino-acid treatments, increased jejunal maltose, lactose and sucrose, and increased ileal sucrose and cellobiose in L-glutamate-fed pigs. These are treatment-associated differences, not direct evidence that the metabolic changes were beneficial. Pathway enrichment identified recurring associations with alanine, aspartate and glutamate metabolism, the citrate cycle, arginine biosynthesis and carbohydrate-related pathways across serum and intestinal samples.

Glutathione metabolism and taurine and hypotaurine metabolism also appeared in intestinal comparisons, while metabolites including glucose-6-phosphate and fructose-6-phosphate were associated with pentose-phosphate-related changes. The analysis used an exploratory false-discovery-rate threshold below 0.2, together with nominal significance, pathway impact above 0.1 and, for selected metabolites, a variable-importance score above 1. The findings are therefore hypothesis-generating: they cannot identify which metabolic route produced an immune difference or show that greater abundance of a metabolite represented improved health.

Several limitations constrain how the results can be interpreted. L-glutamate and L-aspartate were added as crystalline ingredients in place of corn, so the diets differed not only in the presence of these amino acids but also in total amino acid and nitrogen supply. The combined group received twice the supplemental amino-acid amount of either single-ingredient group, making additive, synergistic and greater-substrate effects impossible to separate.

Only six or seven pigs per group were assessed at the terminal tissue-sampling points, limiting precision for segment-specific immune results. The artificial challenge also does not reproduce the full conditions of commercial production. Taken together, the experiment shows that L-glutamate and L-aspartate can modulate selected hematological, inflammatory and metabolic responses during experimental ETEC challenge in weaned piglets, but it does not demonstrate reduced antibiotic need, improved growth or superiority of the combined treatment.

Subject of Research: Effects of L-glutamate and L-aspartate supplementation on hematological, immune and metabolic responses in ETEC-challenged weaned piglets

Article Title: Dietary L-glutamate, L-aspartate, and their combination supplementation modulated hematological, immune, and metabolic responses in weaned piglets challenged with enterotoxigenic Escherichia coli

Article References: Wongchanla, S., Park, Y., Park, S., Sun, S., & Liu, Y. (2026). Dietary L-glutamate, L-aspartate, and their combination supplementation modulated hematological, immune, and metabolic responses in weaned piglets challenged with enterotoxigenic Escherichia coli. Frontiers in Veterinary Science, 13, 1938098. https://doi.org/10.3389/fvets.2026.1938098

Image Credits: AI Generated

DOI: 10.3389/fvets.2026.1938098

Keywords: ETEC, weaned piglets, L-glutamate, L-aspartate, amino acid nutrition, intestinal immunity, hematology, inflammation, metabolomics, piglet nutrition, Dietary, combination

The significance of the work lies in what it replaces. Virtually all industrial fermentation that yields nitrogen-rich products, from amino acids to nucleotides, depends on ammonium salts or urea as the nitrogen feedstock, and those inputs trace back to Haber–Bosch ammonia synthesis. That process, which combines atmospheric nitrogen with hydrogen under extreme pressures and temperatures, consumes an estimated one to two percent of global energy output and generates substantial carbon dioxide emissions as a byproduct of hydrogen production from natural gas. Biological nitrogen fixation, carried out by the nitrogenase enzyme complex in certain bacteria and archaea, performs the same chemical transformation at ambient temperature and pressure using ATP and electrons, offering a potentially far gentler route to usable nitrogen.

Azotobacter vinelandii has long served as the model organism for aerobic nitrogen fixation, a biologically awkward combination since oxygen both damages nitrogenase and competes for the electrons the enzyme needs. The bacterium survives this paradox through extraordinarily high respiratory rates that consume oxygen before it can reach the enzyme. Previous efforts had engineered A. vinelandii to excrete ammonium, the natural next step after nitrogen fixation, since the fixed nitrogen normally feeds the cell’s own biosynthesis. But the researchers behind the new study found that studies on actually using that excreted ammonium as a nitrogen source for other microbes remained limited, and that ammonium production levels were too low to be industrially interesting.

To push yields higher, the team took aim at NifA, the transcriptional activator that switches on the entire nif regulon encoding the nitrogen fixation machinery. They constructed A. vinelandii strains by integrating a nifA overexpression cassette, driven by the strong tac promoter, into the algU locus of the chromosome. This chromosomal integration strategy matters for stability: rather than relying on a plasmid that could be lost during cultivation, the engineered construct is inherited by every daughter cell, ensuring the nitrogen fixation program stays dialed up throughout a fermentation run. The resulting strain stably produced ammonium at a concentration of 1 gram per liter in simple flask cultures, a level the researchers describe as a solid baseline for the platform.

Flask cultures, however, are a proving ground rather than a production environment. The team then scaled the process into a jar fermenter, where they could control oxygen transfer, pH and feeding with far greater precision. By combining fed-batch cultivation, in which fresh carbon source is supplied incrementally to avoid depletion or overflow metabolism, with antifoam treatment to keep the aerated broth from foaming over and lactic acid addition to manage the culture’s chemistry, they raised ammonium production to 1.5 grams per liter. Each of these process interventions addresses a practical bottleneck: fed-batch keeps the energy supply matched to the nitrogenase’s enormous ATP appetite, antifoam protects oxygen transfer and prevents contamination pathways, and acid addition stabilizes the pH as ammonium accumulates and shifts the broth’s acid-base balance.

With a reliable ammonium source in hand, the researchers turned to the second half of the partnership. Corynebacterium glutamicum is arguably the most successful amino acid production organism in industrial biotechnology, responsible for the bulk of the world’s several-million-ton annual L-glutamate output, the flavor-enhancing component of monosodium glutamate. In the co-culture scheme, the engineered A. vinelandii functions as a living nitrogen fertilizer, continuously fixing atmospheric nitrogen gas and releasing ammonium into the shared medium, while C. glutamicum assimilates that ammonium and channels it through its existing metabolic machinery into L-glutamate. The division of labor elegantly sidesteps the need to purify or concentrate the intermediate: the product of one microbe is the substrate of the other, delivered in situ.

The results demonstrated the concept convincingly. Co-cultivation of the ammonium-producing A. vinelandii strain with C. glutamicum enabled the production of 2 grams per liter of L-glutamate from nitrogen gas. While that titer remains well below the tens of grams per liter achieved in conventional glutamate fermentations fed with commercial ammonium, the demonstration establishes a complete biological pipeline from atmospheric dinitrogen to a finished amino acid in a single vessel. The researchers frame the strategy as a contribution to the development of environment-friendly fermentation processes for producing various nitrogen-containing compounds from nitrogen gas, suggesting the platform could extend well beyond glutamate to other amino acids, nucleotides and nitrogenous chemicals.

The engineering choices embedded in the study reveal a careful reading of nitrogenase regulation. NifA sits atop a hierarchy of control mechanisms that bacteria use to avoid wasting energy on nitrogen fixation when fixed nitrogen is already available, a regulatory logic that normally shuts the system down precisely when engineers want it running. By overexpressing NifA from a constitutive tac promoter, the team effectively overrides the ammonium-sensing feedback that would otherwise silence the nif genes as product accumulates. Placing the cassette at the algU locus, which governs stress responses in A. vinelandii, reflects a deliberate choice of a neutral genomic landing site that disrupts native function minimally while granting stable, high-level expression of the activator.

Scaling challenges remain before such co-cultures could challenge conventional plants. Nitrogenase is an enzyme of notorious fragility and metabolic cost, demanding roughly sixteen ATP per molecule of nitrogen reduced, and maintaining two microbial populations with different physiological optima in one fermenter requires balancing oxygen availability, carbon source preference and growth rates. The fed-batch jar fermenter results, with their combination of antifoam and lactic acid management, hint at the kind of process engineering refinement that will determine whether titers can climb toward commercial relevance. The involvement of Kikkoman Corporation, a company with deep roots in fermentation technology, alongside academic groups at The University of Tokyo’s Department of Biotechnology and Collaborative Research Institute for Innovative Microbiology, suggests industrial interest in closing that gap. Several of the authors have filed patent applications on the work, underscoring its perceived commercial potential.

For now, the study stands as a proof of concept with a compelling narrative: a flavor compound that seasons much of the world’s food, assembled in part from the air itself, by two bacteria cooperating in a fermenter. If the platform’s titers can be improved through further strain and process optimization, nitrogen-fixing co-cultures could offer fermentation industries a route to decouple amino acid production from synthetic fertilizer inputs, trimming both energy demand and carbon emissions. The researchers position their work as a step toward fermentation processes that draw their nitrogen directly from the atmosphere, converting a century-old industrial dependency into a biological partnership.

Subject of Research: L-glutamate production from nitrogen gas via co-culture of engineered Azotobacter vinelandii and Corynebacterium glutamicum

Article Title: L-Glutamate production from nitrogen gas by co-culturing Corynebacterium glutamicum with Azotobacter vinelandii

Article References: Ito, Y., Yoshidome, D., Araki, Y., Ito, K., Hidaka, M., Kosono, S., & Nishiyama, M. (2026). L-Glutamate production from nitrogen gas by co-culturing Corynebacterium glutamicum with Azotobacter vinelandii. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-026-14031-5

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14031-5

Keywords: Azotobacter vinelandii, Corynebacterium glutamicum, nitrogen fixation, L-glutamate, co-culture, nifA overexpression, ammonium production, Haber-Bosch, metabolic engineering, fed-batch fermentation, nitrogenase, sustainable biotechnology